A specific D-glucose-inhibitable cytochalasin B binding assay for quantitating the number of glucose transport systems in subcellular membrane fractions has previously been used to demonstrate that insulin stimulates glucose transport in the isolated rat adipose cell primarily through the translocation of glucose transport systems from a large intracellular pool to the plasma membrane. This intracellular pool is associated with the cell's low-density microsomal membranes. This assay has now been used to demonstrate that this translocation process in the rat adipose cell is multi-stepped, extremely rapid, completely reversible, and insulin concentration-dependent. In addition, insulin appears to stimulate glucose transport in rat diaphragm and the isolated human adipose cell through this same translocation mechanism. Furthermore, the rat adipose cell's intracellular glucose transport systems appear to be 1) localized in either a unique subfraction of the Golgi apparatus or a distinct membrane species, 2) immunologically similar to the human erythrocyte glucose transporter with a MW of roughly 45,000 daltons, and 3) chemically processed during their cycling to the plasma membrane in response to insulin.